The present invention is directed toward the development of an oleaginous yeast that accumulates oils enriched in long-chain ω-3 and/or ω-6 polyunsaturated fatty acids (“PUFAs”; e.g., 18:3, 18:4, 20:3, 20:4, 20:5, 22:6 fatty acids). Thus, in addition to developing techniques to introduce the appropriate fatty acid desaturases and elongases into these particular host organisms (where naturally produced PUFAs are usually limited to production of 18:2 fatty acids [and less commonly, 18:3 fatty acids]), it is also necessary to increase the transfer of PUFAs into storage lipid pools following their synthesis.
Most free fatty acids become esterified to coenzyme A (CoA), to yield acyl-CoAs. These molecules are then substrates for glycerolipid synthesis in the endoplasmic reticulum of the cell, where phosphatidic acid and diacylglycerol (DAG) are produced. Either of these metabolic intermediates may be directed to membrane phospholipids (e.g., phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) or DAG may be directed to form triacylglycerols (TAGs), the primary storage reserve of lipids in eukaryotic cells.
In the yeast Saccharomyces cerevisiae, three pathways have been described for the synthesis of TAGs. First, TAGs are mainly synthesized from DAG and acyl-CoAs by the activity of diacylglycerol acyltransferases.
More recently, however, a phospholipid:diacylglycerol acyltransferase has also been identified that is responsible for conversion of phospholipid and DAG to lysophospholipid and TAG, respectively, thus producing TAG via an acyl-CoA-independent mechanism (Dahlqvist et al., PNAS. 97(12):6487-6492 (2000)). Finally, two acyl-CoA:sterol-acyltransferases are known that utilize acyl-CoAs and sterols to produce sterol esters (and TAGs in low quantities; see Sandager et al., Biochem. Soc. Trans. 28(6):700-702 (2000)).
A comprehensive mini-review on TAG biosynthesis in yeast, including details concerning the genes involved and the metabolic intermediates that lead to TAG synthesis, is that of D. Sorger and G. Daum (Appl. Microbiol. Biotechnol. 61:289-299 (2003)). However, the authors acknowledge that most work performed thus far has focused on Saccharomyces cerevisiae and numerous questions regarding TAG formation and regulation remain. In this organism it has been conclusively demonstrated that only four genes are involved in storage lipid synthesis: ARE1 and ARE2 (encoding acyl-CoA:sterol-acyltransferases), LRO1 (encoding a phospholipid:diacylglycerol acyltransferase, or PDAT enzyme) and DGA1 (encoding a diacylglycerol acyltransferase, or DGAT2 enzyme) (Sandager, L. et al., J. Biol. Chem. 277(8):6478-6482 (2002)). Homologs of these genes have been identified in various other organisms and disclosed in the public literature, but none of these genes have been isolated from oleaginous yeast. Furthermore, techniques for modifying the transfer of fatty acids to the TAG pool in oleaginous yeast have not been developed. Thus, there is a need for the identification and isolation of genes encoding acyltransferases that will be suitable for use in the production and accumulation of PUFAs in the storage lipid pools (i.e., TAG fraction) of oleaginous yeast.
Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oleaginous yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica). These organisms can accumulate oil up to 80% of their dry cell weight; and, the technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)). Most recently, the natural abilities of oleaginous yeast (mostly limited to 18:2 fatty acid production) have been enhanced by advances in genetic engineering, leading to the production of 20:4 (arachidonic acid), 20:5 (eicosapentaenoic acid) and 22:6 (docosahexaenoic acid) PUFAs in transformant Yarrowia lipolytica. These ω-3 and ω-6 fatty acids were produced by introducing and expressing heterologous genes encoding the ω-3/ω-6 biosynthetic pathway in the oleaginous host (see co-pending U.S. application Ser. No. 10/840,579).
The importance of PUFAs are undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA) or α-linolenic acid (ALA); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or TAGs; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al., Lancet 2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., and Dyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). And, numerous other studies-document wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 fatty acids against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
PUFAs are generally divided into two major classes (consisting of the ω-6 and the ω-3 fatty acids) that are derived by desaturation and elongation of the essential fatty acids, LA and ALA, respectively. Despite a variety of commercial sources of PUFAs from natural sources [e.g., seeds of evening primrose, borage and black currants; filamentous fungi (Mortierella), Porphyridium (red alga), fish oils and marine plankton (Cyclotella, Nitzschia, Crypthecodinium)], there are several disadvantages associated with these methods of production (e.g., highly heterogeneous oil compositions, accumulation of environmental pollutants, uncontrollable fluctuations in availability due to weather/disease, expense at the commercial scale). As a result of these limitations, extensive work has been conducted toward: 1.) the development of recombinant sources of PUFAs that are easy to produce commercially; and 2.) modification of fatty acid biosynthetic pathways, to enable production of desired PUFAs. Advances in the isolation, cloning and manipulation of fatty acid desaturase and elongase genes from various organisms have been made over the last several years. Knowledge of these gene sequences offers the prospect of producing a desired fatty acid and/or fatty acid composition in novel host organisms that do not naturally produce PUFAs.
As described in Picataggio et al. (co-pending U.S. patent application Ser. No. 10/840,579), oleaginous yeast have been identified as an appropriate microbial system in which to express PUFA desaturase and elongase genes to enable economical production of commercial quantities of one or more PUFAs in these particular hosts. To further advance the work described therein towards the development of an oleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 fatty acids, however, it is necessary to increase the transfer of these PUFAs into storage TAGs (oil), once they are synthesized by fatty acid desaturases and elongases. Thus, there is a need for the identification and isolation of genes encoding acyltransferases that will be suitable for use in the production and accumulation of PUFAs in TAGs. Techniques for modifying the transfer of fatty acids to the TAG pool in oleaginous yeasts must also be developed.
Applicants have solved the stated problem by isolating the genes encoding PDAT and DGAT2 from the oleaginous yeast, Yarrowia lipolytica. These genes will be useful to enable one to modify the transfer of free fatty acids (e.g., ω-3 and/or ω-6 fatty acids) to the TAG pool in oleaginous yeast.